The present invention relates to methods of treating and preventing cancer, viral infection, or microbial infection in an animal comprising administrating to said animal antibodies specific for C3b(i). The present invention also relates to methods of treating and preventing cancer, viral infection, or microbial infection in an animal comprising administering to said animal IgG antibodies, IgM antibodies and/or complement components in combination with antibodies immunospecific for C3b(i). The present invention also relates methods of treating and preventing cancer, viral infection or microbial infection in an animal comprising administering to said animal antibodies that immunospecifically bind to one or more cancer cell antigens, viral antigens or microbial antigens, respectively, in combination with antibodies immunospecific for C3b(i). The present invention also relates to pharmaceutical compositions for the treatment and prevention of cancer, viral infection, and microbial infection comprising antibodies immunospecific for C3b(i). Further, the present invention relates to the detection, imaging, and diagnosis of cancer utilizing antibodies immunospecific for C3b(i).
The complement system which is composed of some 21 plasma proteins plays an important role in the human immune system, both in the resistance to infections and in the pathogenesis of tissue injury. The activated products of the complement system attract phagocytic cells and greatly facilitate the uptake and destruction of foreign particles by opsonization. There are two distinct pathways for activating complement, the classical pathway and the alternate pathway, that result in conversion of C3 to C3b and subsequent responses (e.g., the formation of the membrane attack complex (xe2x80x9cMACxe2x80x9d)). Activation of the classical pathway is initiated by antigen-antibody complexes or by antibody bound to cellular or particulate antigens. The alternate pathway is activated independent of antibody by complex polysaccharides in pathogens such as bacterial wall constituents, bacterial lipopolysaccharides (LPS) and cell wall constituents of yeast (zymosan).
The classic complement pathway is initiated by the binding of C1 to immune complexes containing IgG or IgM antibodies. Activated C1 cleaves C2 and C4 into active components, C2a and C4b. The C4b2a complex is an active protease called C3 convertase, and acts to cleave C3 into C3a and C3b. C3b forms a complex with C4b2a to produce C4b2a3b, which cleaves C5 into C5a and C5b. C5b combines with C6, and the C5b6 complex combines with C7 to form the ternary complex C5b67. The C5b67 complex binds C8 to form the C5b678 complex which in turn binds C9 and results in the generation of the C5-C9 MAC. The insertion of the MAC into the cell membrane results the formation of a transmembrane channel that causes cell lysis.
In the alternative pathway, conversion of C3 to C3b (or C3i) produces a product that can combine with factor B, giving C3bB (or C3iB). These complexes are acted upon by factor D to generate C3bBb, which is a C3 convertase capable of cleaving more C3 to C3b, leading to more C3bBb and even more C3 conversion. Under certain circumstances the C3bBb complex is stabilized by association with the positive regulator properdin (P) by association of C3b and Bb. The C3 convertases can associate with an additional C3b subunit to form the C5 convertase, C3bBb C3b, which is active in the production of the C5-C9 MAC.
In both the classical and alternative pathways, the critical step in the activation of complement is the proteolytic conversion of C3 to the fragments C3b and C3a. C3a is an anaphylatoxin that attracts mast cells to the site of challenge, resulting in local release of histamine, vasodilation and other inflammatory effects. The nascent C3b has an ability to bind to surfaces around its site of generation and functions as a ligand for C3 receptors mediating, for example, phagocytosis.
Endogenous cell surfaces normally exposed to complement are protected by membrane-bound regulators such as decay accelerating factor (xe2x80x9cDAFxe2x80x9d), C59 (xe2x80x9cprotectinxe2x80x9d), MCP, and the soluble C1 inhibitor or C1NH. DAF and MCP are responsible for limiting production of C3b and insure the generation of inactive forms of C3b, C3bi and C3dg from C3b. CD59 prevents attack of the MAC, which would otherwise destroy the cancer cell. C1 inhibitor binds to the active subcomponents of C1, C1r and C1s, and inhibits their activity.
Despite advances in prevention and early detection, refinements in surgical technique, and improvements in adjuvant radiotherapy and chemotherapy, the ability to cure many patients of cancer remains elusive. This is especially pertinent to prostate cancer, which remains the most prevalent visceral tumor in American men, with approximately 180,000 new cases and nearly 40,000 deaths expected in 1999 (Landis et al., 1999, Cancer J Clin 49: 8-31). The continuing challenge of prostate cancer treatment is the successful management and eradication of recurrent, metastatic, and hormone-refractory disease, which accounts for the vast majority of prostate cancer-specific morbidity and mortality (Small, 1998, Drugs and Aging 13:71-81).
Many treatment modalities currently under investigation for prostate and other cancers depend upon tissue-specific delivery of anti-neoplastic agents. One immunotherapeutic approach involves conjugating cytotoxic agents to monoclonal antibodies (mAbs) specific for a particular cancer cell epitope. In this manner, the therapeutic agents can be delivered at a high therapeutic dose directly, and selectively, to the tumor site, thereby minimizing injury to healthy tissue (Bach et al., 1993, Immunol Today 14:421-5; Reithmuller et al., 1993, Cur. Op. Immunol 5:732-9; and Gruber et al., 1996, Spring Sem Immunopath 18:243-51). This method first requires the identification of specific epitopes for each cancer type. Such candidate epitopes must be expressed at high levels on the cancer cells compared to normal tissue. Second, this method requires the development of high affinity mAbs specific for these epitopes and these mAbs must show minimal cross-reactivity with self tissue. The biological mechanism of killing with mAbs will be variable, depending upon the epitopes identified on the cancer cells, and the effector functions of the specific mAb isotype. However, due to antigenic modulation and/or mutation, the cancer cells may reduce the available levels of the target epitope per cell, or eliminate it from their surface altogether. Thus, the use of mAbs in cancer diagnosis and treatment remains problematic.
A more widely applicable approach to treatment of cancer with mAbs would be to identify a ubiquitous antigenic site, present on virtually all cancer cells, and then to develop a panel of mAbs specific for this antigen. A voluminous literature reveals that cancer cells share certain common characteristics. Many types of human cancer cells are characterized by substantial abnormalities in the glycosylation patterns of their cell-surface proteins and lipids (Hakomori et. al., 1996, Canc Res. 56:5309-18; Castronovo et al., 1989, J Nat Canc Inst 81:212-6; Springer et al., 1984, Science 224:1198-206; and Springer et al., 1997, J Mol Med 75:594-602). These differences have led to the identification of antigenic determinants on cancer cells which are expressed at far lower levels on normal cells. Natural IgM antibodies to these epitopes are present in the circulation, and the interaction of such IgM antibodies with these cancer cell surface antigens leads to activation of complement and covalent coupling of complement activation products (C3b and its fragments, collectively referred to as C3b(i)) to the tumor cells (Okada et al., 1974, Nature 248:521-25; Irie et. al., 1974, Science 186:454-456; Desai et al., 1995, J Immunol Methods 188:175-85; Vetvicka et al., 1996, J Clin Invest 98:50-61; Vetvicka et al., 1997, J Immunol 159:599-605; and Vetvicka et al., 1999, Clin Exp Immunol 115:229-35). Although relatively large amounts of C3b(i) can be deposited on cancer cells, the concomitant expression of high levels of membrane-associated complement control proteins (e.g., decay accelerating factor (xe2x80x9cDAFxe2x80x9d), membrane cofactor protein (xe2x80x9cMCPxe2x80x9d), and, in particular, xe2x80x9cprotectinxe2x80x9d i.e., CD59) usually prevents complement-mediated lysis (Cheung et al., 1988, J Clin Invest 81:1122-8; Gorter et al., 1996, Lab Invest 74:1039-49; Maenpaa et al., 1996, Am J Path 148:1139-52; and Li et al., 1997, Int J Canc 71:1049-55). Further, several investigators have established that in most cases, cancer patients have substantially lowered levels of the potentially protective IgM antibodies. Thus, in many cases the cancer cells cannot easily be killed by complement activation because of the reduced levels of protective IgM antibody and the increased expression of human complement control proteins on their surface.
Innate immunity allows humans and most animals to respond to foreign organisms even before the initiation of the immune response. One of the most important aspects of innate immunity involves the complement system (Cooper, N. R. Complement and viruses. Volanakis, J. B. and Frank, M. M. The Human Complement System in Health and Disease. 18, 393-408. (1998) New York, Marcel Dekker, Inc.; and Petry, F. and Loos, M. Bacteria and complement. Volanakis, I. E. and Frank, M. M. The Human Complement System in Health and Disease. 171, 375-392. (1998) New York, Marcel Dekker, Inc.). It is now well-documented that a wide variety of bacteria, viruses and other microorganisms activate complement in the immunologically naive individual (Peterson et al., 1978, Infect. Immun. 19:943; Verbrugh et al., 1982, J. Immunol. 129:1681; Newman et al., 1985, J. Exp. Med. 161:1414; Seelen et al., 1995, Immunol. 84:653; Wagner et al., 1998, J. Immunol. 160:1936; Bakker et al., 1992, AIDS 6:35; Hiavacek et al., 1999, Proc. Nat!. Acad. Sci. 96:14681; Schmitz et al., 1994, J. Immunol. 153:1352; Tacnet-Delorme et al., 1999, J. Immunol. 162:4088; Joling et al., 1993, J. Immunol. 150:1065; Dominguez et al., 1999, J. Exp. Med. 189:25; and Washburn et al., 1991, Molec. Immunol. 28:465). These include, but are not limited to, both gram positive and gram negative bacteria, and the virus which causes AIDS, HIV. As a consequence of complement activation, these organisms are covalently labeled with complement activation fragments, and in particular with C3b(i). Moreover, in individuals with previous exposure to these pathogens (either through immunization or previous infection), specific antibodies will bind to the pathogens and also activate complement and promote C3b(i) deposition. Although in many instances this complement activation ultimately leads to the clearance and destruction of the microorganism, in some cases the C3b(i)-labeled invader remains in the circulation and can still infect susceptible tissues and organs. In fact, there are many examples of microorganisms which actually use these covalently attached C3b(i) molecules to gain entry into cells (which have receptors for C3bCi)) and establish productive infections (Bakker et al., 1992, AIDS 6:35; Tacnet-Delorme et al., 1999, J. Immunol. 162:4088; Cooper, N. R. Complement and viruses. Volanakis, J. B. and Frank, M. M. The Human Complement System in Health and Disease. 18, 393-408. 1998. New York, Marcel Dekker, Inc.; and Petry, F. and Loos, M. Bacteria and complement. Volanakis, I. E. and Frank, M. M. The Human Complement System in Health and Disease. 171, 375-392. (1998) New York, Marcel Dekker, Inc.). Several lines of evidence indicate that C3b(i)-opsonized HIV can persist in the body bound to the surface of dendritic cells for very long periods of time, and this cell-bound HIV may represent one of the main obstacles to the permanent elimination of HIV from the body (Hiavacek et al., 1999, Proc. Nat!. Acad. Sci. 96:14681; and Schmitz, et al., 1994,. J. Immunol. 153:1352).
Citation of a reference in this section or any section of this application shall not be construed as an admission that such reference is prior art to the present invention.
The present invention encompasses compositions comprising antibodies immunospecific for C3b(i) and methods for the treatment and prevention of cancer, viral infection, and microbial infection in an animal comprising administering such compositions to said animal. In particular, the present invention provides methods for treating or preventing cancer, viral infection, and microbial infection in animal comprising administering to said animal a therapeutically or prophylactically effective amount of an antibody immunospecific for C3b(i), an antibody immunospecific for C3b(i) covalently linked to a second molecule (e.g., an IgM antibody and IgG antibody, a glycoprotein or a glycolipid), a nucleic acid sequence encoding an antibody immunospecific for C3b(i), or a nucleic acid sequence encoding an antibody immunospecific for C3b(i) covalently linked to a second molecule. The present invention also provides methods for the treatment and prevention of cancer, viral infection, and microbial infection in an animal comprising administering to said animal IgG antibodies, IgM antibodies and/or complement components in combination with anti-C3b(i) antibodies. The present invention also provides methods for the treatment and prevention of cancer, viral infection, and microbial infection in an animal comprising administering to said animal antibodies immunospecific for one or more cancer cell antigens, viral antigens, or microbial antigens, respectively, in combination with anti-C3b(i) antibodies. The present invention further provides methods for depleting cancer cells from cells obtained from an animal with cancer comprising contacting in vitro a sample comprising cells obtained from said animal with one or more antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule.
The present invention also provides pharmaceutical compositions comprising one or more antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule, in an amount effective for the treatment or prevention of cancer, a viral infection, or a microbial infection in an animal. The present invention also provides pharmaceutical compositions comprising one or more nucleic acid molecules encoding one or more antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule, in an amount effective for the treatment or prevention of cancer, viral infection, or microbial infection in an animal. The present invention further provides a pharmaceutical composition comprising a bispecific antibody which is immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule and an effector cell receptor or antigen, in an amount effective for the treatment or prevention of cancer, viral infection, or microbial infection in an animal.
The present invention encompasses methods for the detection, imaging, and diagnosis of cancer utilizing antibodies or fragments thereof immunospecific for C3b(i). The present invention provides methods for detecting cancer in an animal comprising: a) administering to said animal an effective amount of a labeled antibody that immunospecifically binds to C3b(i) or C3b(i) covalently linked to a second molecule; b) waiting for a time interval following the administering to permit the labeled antibody to preferentially concentrate at any cancerous site in the subject; c) determining background level; and d) detecting the labeled antibody in the animal, wherein detection of the labeled antibody above the background level indicates the presence of a cancer. The present invention also provides a method for detecting cancer in an animal, comprising imaging said subject at a time interval after administrating to said animal of an effective amount of a labeled antibody that immunospecifically binds to C3b(i) or C3b(i) covalently linked to a second molecule, said time interval being sufficient to permit the labeled antibody to preferentially concentrate at any cancerous site in said subject, wherein detection of the labeled antibody localized at said site in the animal indicates the presence of cancer.
The present invention provides kits comprising, in one or more containers, an antibody immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule with instructions for use in the detection, imaging or diagnosis of cancer.
The term xe2x80x9cC3b(i)xe2x80x9d as used herein refers to C3b and its fragments, including, but not limited to, C3b(i), C3b, and C3d.
As used herein, reference to an antibody immunospecific for C3b(i), C3b(i) specific antibodies, or anti-C3b(i) antibodies and the like shall be construed as including one or more antibodies immunospecific for C3b(i) covalently linked to a second molecule, unless indicated otherwise explicitly or by context. In a preferred embodiment, an anti-C3b(i) antibodies preferentially bind to C3b-opsonized cancer cells, and not C3b(i) in the mileau. In another preferred embodiment, anti-C3b(i) antibodies preferably bind to C3b(i) deposited on viruses or microbes, and not C3b(i) in the mileau.
The term xe2x80x9cfusion proteinxe2x80x9d as used herein refers to a polypeptide that comprises an amino acid sequence of an antibody or fragment thereof and an amino acid sequence of a heterologous polypeptide (i.e., an unrelated polypeptide).
The term xe2x80x9chost cellxe2x80x9d as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d antibody or fragment thereof, or polypeptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language xe2x80x9csubstantially free of cellular materialxe2x80x9d includes preparations of an antibody, an antibody fragment, or a polypeptide in which the antibody, antibody fragment or polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody, an antibody fragment or a polypeptide that is substantially free of cellular material includes preparations of antibody, antibody fragment or polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a xe2x80x9ccontaminating proteinxe2x80x9d). When the antibody, antibody fragment, or polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the antibody, antibody fragment or polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the antibody, antibody fragment, or polypeptide have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody, or polypeptide fragment of interest. In a preferred embodiment, antibodies of the invention or fragments thereof are isolated or purified.
An xe2x80x9cisolatedxe2x80x9d nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an xe2x80x9cisolatedxe2x80x9d nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a preferred embodiment, nucleic acid molecules encoding antibodies of the invention or fragments thereof are isolated or purified.
FIG. 1 (A-D). Representative flow cytometry data from a study with serum from a normal donor (A, B) and a cancer patient (C, D). Measurement of C3b(i) (A, C) and IgM (B, D) deposition on C4-2 human prostate cancer cells is shown. Abundant C3b(i) is deposited on C4-2 cancer cells in response to the addition of normal human serum; this opsonization appears to be facilitated by both the classical and alternative complement pathways. After opsonization with serum from a prostate cancer patient, significantly less C3b(i) and IgM are deposited on the tumor cells (C, D). C3b(i) deposition via the alternative pathway (serum with Mg-EGTA), however, is comparable for both the normal and cancer patient serum, suggesting that the alternative pathway of the complement system remains intact in prostate cancer patient serum.
FIG. 2 (A-B). Flow cytometry (A) and radioimmunoassay (B) data demonstrating that removal of IgM from AB-positive serum results in a large reduction in the amount of C3b(i) that is deposited on LNCaP (A) or C4-2 (B) cells. C3b(i) deposition can be erstored with either whole normal human plasma (A, B) (e.g., plasma/IgM-depleted serum), whcih provides a source of human IgM, or with purified IgM/IgM-depleted serum (B).
FIG. 3 (A-B). Radioimmunoassay data demonstrating that complement activation generates between 50,000 and 500,000 C3b(i) epitopes/opsonized cancer cell (net binding, background subtracted), as defined by binding of both 125I-labeled mAb 7C12 and 3E7. In panel A, an AB-positive serum was used for opsonization and, after three washes, cells were probed with differing amounts of the two mAb. Alternatively, mAb 3E7 was added to the cells just before the serum, and was present during opsonization. In panel B, AB-positive serum was used in conjunction with the 1-xcexcg/ml and 10-xcexcg/ml probes, and an AB-positive citrated plasma (different donor from that in A) was used for the 3-xcexcg/ml probes
FIG. 4. Flow cytometry results from surveys of sera from normal donors and patients with prostate cancer. Binding of human immunoglobulin to LNCaP and C4-2 prostate cancer cells was measured. Significant differences were determined by t-tests.
FIG. 5 (A-B). Immunohistochemical staining of normal (A) and neoplastic (B) human prostate tissue after incubation with anti-C3b(i) mAb.
FIG. 6. Rosetting experiment using erythrocytes and opsonized LNCaP prostate cancer cells incubated in plasma in the presence of an anti-CR1 X anti-C3b(i) bispecific monoclonal antibody complex (7G9 X 3E7).
FIG. 7 (A-B). In vitro killing of LNCaP (A) and C4-2 (B) prostate cancer cells using 131I-labeled mAbs. Dashed line ( - - - ) delineates normal serum opsonized cells treated with 131I-labeled irrelevant mAbs; dotted line ( . . . ) delineates non-opsonized cells treated with 131I-anti-Cb3(i) mabs; solid line (xe2x80x94) delineates normal serum opsonized cells treated with 131I-labeled anti-C3b(i) mAbs. Measured as cell proliferation relative to non-treated cells.
FIG. 8. The schematic illustrates the steps of the invention, all of which occur on the cell surface of tumor cells within the body of the cancer patient. In the first step, human IgM (either endogenous, or infused into the patient) binds to specific sites on the cancer cell. In the second step, complement (either endogenous, or infused into the patient as fresh plasma) is activated, and the resulting proteolytic fragment C3b(i) is deposited on the surface of the cancer cell. In the third step, a mAb specific for the C3b(i) epitope is administered. The mAb can be associated with a toxic, enzymatic, genetic, differentiating, and/or imaging agent (therefore it is an xe2x80x9ceffector mAbxe2x80x9d), which results in the destruction or imaging of the cancer cell.
FIG. 9. Red cell binding experiment using erythrocytes and opsonized Rajii cells. Rajii cells treated with: serum alone; EDTA treated serum alone; serum and Rituximab; EDTA treated serum and Rituximab; serum and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes; EDTA treated serum and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes; serum, Rituximab, and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes; and EDTA treated serum Rituximab, and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes.
FIG. 10. Red cell binding experiment using erythrocytes and opsonized Rajii cells. Rajii cells treated with: washed whole blood reconstituted serum alone; washed whole blood reconstituted serum and Rituximab; washed whole blood reconstituted serum and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes; and washed whole blood reconstituted serum, Rituximab, and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes; washed whole blood reconstituted serum, Rituximab (15xe2x80x2), and anti-C3b(i) X anti-CR1 bispecific monoclonal antibody complexes; and washed whole blood reconstituted serum, anti-C3b(i) X anti-CR1 bispecific monoclonal antibody (15xe2x80x2), and Rituximab.